Preparation of Tc99m-Labeled Pseudomonas Bacteriophage without

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Preparation of Tc99m-labeled Pseudomonas Bacteriophage without Adversely Impacting Infectivity or Biodistribution. Derek Holman, Matthew P Lungren, Jonathan Hardy, Christopher H. Contag, and Francis G. Blankenberg Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00395 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Bioconjugate Chemistry

Preparation of Tc99m-labeled Pseudomonas Bacteriophage without Adversely Impacting Infectivity or Biodistribution. Derek Holman†, Matthew P. Lungren‡, Jonathan Hardy§, Chris Contag†,‡, §, Francis Blankenberg‡* †

Department of Pediatrics, Division of Medical Genetics and Biochemistry, Stanford

University School of Medicine, Stanford, CA 94305, USA ‡

Department of Radiology, Division of Pediatric Radiology and Nuclear Medicine, Lucile

Packard Children’s Hospital, Stanford, CA 94305, USA §

Department of Microbiology and Immunology, E150 Clark Center MC 5427, Stanford

University School of Medicine, Stanford, CA 94305, USA *Communicating Author- Francis G. Blankenberg, M.D. e-mail: [email protected] Abstract Bacteriophages (phages) are ubiquitous viruses which have adapted to infect and replicate within target bacteria, their only known hosts, in a strain specific fashion with minimal cross infectivity. The recent steep rise in antibiotic resistance throughout the world has renewed interest in adapting phages for the imaging and treatment of bacterial infection in humans. In this article, we describe the current limitations surrounding the radiolabeling of phage for the imaging and treatment of bacterial infection and methods to overcome these difficulties. Specifically, we examined the effects of hydrazinonicotinamide conjugation and removal of bacterial DNA on the infectivity, biodistribution and radionuclide imaging of a phage lytic for a clinically relevant strain of Pseudomonas aeruginosa, a common gram negative bacterial pathogen often resistant to multiple antibiotics. We found that all but the briefest reaction of concentrated phage with hydrazinonicotinamide (≤ 3 minutes) resulted in nearly complete loss of infectivity. Furthermore, we determined that digestion and removal of bacterial DNA was needed to avoid high non-specific uptake of hydrazinonicotinamide-labeled phage within the liver and spleen as well as prolonged circulation in the blood. We also demonstrate the surprisingly wide soft tissue and organ biodistribution and rapid pharmacokinetics of

99m

Tc-hydrazinonicotinamide-labeled phage

in normal mice as well as its imaging characteristics and efficacy in wounded mice infected with bioluminescent Pseudomonas aeruginosa. In conclusion, the preservation of phage infectivity and removal of all bacterial containments including DNA are critical methodologic considerations in the labeling of phages for imaging and therapy. ACS Paragon Plus Environment


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Introduction The increasing threat of antibiotic-resistance has re-ignited interest in the use of bacteriophages (phages), natural predators of bacteria, for the treatment of multi-drug resistant bacterial wound and urinary infections as well as osteomyelitis, otitis media and sepsis1-13. Despite, human exposure to phages from birth and continuously throughout life, the safety and efficacy of phage preparations for use in controlling bacterial infections in humans beyond the skin and external ear are currently unknown14,15. As part of efforts to better understand the fate of phages within the body, multiple investigators have examined the biodistribution of administered phage in animal models or human samples either by plaque assay of homogenized samples of organs and tissues of interest16-24 or by radiolabeling phage for radionuclide imaging and tracer uptake studies25-30. Several recent publications have also suggested the potential of radiolabeled phage as a specific imaging marker of bacterial infection in a variety of animal models27-30. The issues of loss of labeled phage infectivity (via plaque assay), high liver and spleen tracer uptake as well as prolonged blood clearance however, have not been adequately addressed in the literature25-30. The goal of our current study was to develop a radiolabeling technique which avoids 1) the inactivation of phage infectivity and 2) the high non-specific hepatic and splenic uptakes associated with prolonged circulation times of intravenously injected radiolabeled material described in previous publications25-30. To this end, we have developed methods to obtain a high titer of purified phage (1015 to 1016 particles/mL) conjugated with hydrazinonicotinamide (HYNIC), a well described bifunctional chelator designed for radiolabeling proteins and peptides with Technetium 99m (99mTc)31-34. We also demonstrate that removing bacterial host DNA from the phage preparation is a critical factor prior to derivatization with HYNIC. Lastly, we show that only brief reactions (≤ 3 minutes at RT) with HYNIC prevent inactivation of phage infectivity. Both DNA removal and brief HYNIC/phage reaction times have profound impacts on the biodistribution, as compared to that found in prior literature and the infectivity of PAML-31-1 phage towards the susceptible to the Pseudomonas aeruginosa strain Xen5, both in vitro and in vivo. Results and Discussion Transmission Electron Microscopy

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Freshly prepared phage prior to HYNIC conjugation underwent transmission electron microscopic imaging at the Cell Science Imaging Facility (Beckman Center at Stanford). Low and high magnification images show that the purified phage exists in two forms: both have non-elongated icosahedral heads approximately 60 nm in diameter, and each has a tail of 100 or 200 nm respectively. Given that the second population had tails that were both longer and thinner, and capsids that, being opaque, are likely filled, we reasoned that the first population represents the contracted, ghost phage post-ejection, and the second population represents the intact phage (Figures 1A & 1B)35. For many of the virions, structures at the base of the tail that could be tail fibers are visible.

A)

B)

Figures 1A & B. Transmission Electron Microscopy of Purified Phage. TEM images of the PAML-31-1 bacteriophage at low (A) and high (B) resolution show the presence of an icosahedral head, a long tail, and an absence of tail fibers. The icosahedral head is approximately 50 nm in diameter, and the tail between 100 and 200 nm in length, depending on tail contraction status. PAML31-1 Genome Annotation and Classification Sequencing and genome assembly determined that phage PAML31-1 has a genome of 65.1Kb, with a GC percentage of 55.37%, that contains 93 different coding sequences. Of these coding sequences, 3 were identified as tail proteins, 1 as a plate protein, 2 as tail fiber proteins, and 3 as head proteins (Figure 2). As evaluated by homology via BLASTn, the PAML31-1 genome sequence was 99% identical to that of Pseudomonas phage NP3, a Pbunavirus in the Myoviridae family. Unlike many other Myoviruses, the Pbunavirus has only 4 tail fibers, each of which protrudes radially in a single plane from the tail plate, explaining why the tail fibers were not easily visible in the electron microscopy images. ACS Paragon Plus Environment


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Figure 2. PAML 31-1 Phage Classification and Genetic Elements. BPhage sequencing and genomic assembly were performed by Sequetech. Phage genome annotation was performed using PHAST (http://phast.wishartlab.com/), and clustering was performed using BLASTn.

Effects of DNase I Treatment and Length of Incubation with HYNIC on Radiolabeling and Biodistribution Our initial experiments were conducted in normal young adult male CD-1 mice using fresh phage without the digestion and removal of bacterial DNA prior to purification and a 2 hour reaction at RT of 3.23 x 1013 phage particles in 300 µL of 20 mM HEPES/1x PBS buffer with 2 µL 40 mM HYNIC/DMF (hydrazinonicotinamide / dimethylformamide); a widely used highly stable bifunctional chelator with one moiety containing Nhydroxysuccinimide (NHS), an active ester which reacts with accessible amine residues31-34. HYNIC-phage was then radiolabeled with the addition of freshly eluted Na-(99mTc)pertechnetate and tin-tricine buffer which permits the second moiety of HYNIC to complete coordination sphere of technetium using tricine as an additional co-ligand36. After 60 minutes, the reaction mixture (Prep#1) was purified on a PD-10 desalting column eluted with ten 1 mL fractions of 1x PBS. While overall labeling efficiency was 96% (i.e. cumulative eluted activity normalized to the sum of residual column and cumulative eluted activity) only 70% of the total 99m

Tc activity was collected in the first 5 fractions while 26% was collected in the subsequent 5 fractions. 100

µL of fraction 3 was intravenously injected into groups of 4 normal mice which were euthanized for biodistribution assay after one hour as shown in Table 1. Similar to previous studies with HYNIC and other forms chemically modified phage, there was high uptake of tracer in the liver, spleen and blood25-30. Prep#1 ACS Paragon Plus Environment

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however, showed nearly complete loss of infectivity (< 1%) as compared with unlabeled phage by in vitro agarose plate serial dilution assay.

Table 1. Biodistribution at 60 minutes (%ID/g)

blood lungs liver spleen stomach kidney

Prep #1 10.7 ± 2.6 2.3 ± 1.0 14.5 ± 7.3 28.5 ± 7.8 2.4 ± 0.4 3.1 ± 1.6

Prep #2 3.5 ± 0.32 1.6 ± 0.11 2.5 ± 0.57 0.9 ± 2.3 1.5 ± 0.45 15.3 ± 3.3

Prep #3 2.1 ± 0.34 0.9 ± 0.27 2.3 ± 0.86 2.1 ± 0.62 0.45 ± 0.14 12.4 ± 0.79

Table 1. Comparison of DNase I Treatment and HYNIC Reaction Time on Biodistribution. Results of the biodistribution of phage one hour after intravenous injection conjugated and radiolabeled with the Prep#1, Prep#2, or Prep#3 protocols are shown in Table 1. Results are expressed as the average of %ID/g (% of Injected Dose activity per gram of tissue) ± one standard deviation with four young adult CD-1 male mice per group. Prep#1: 2 hr HYNIC conjugation, 2 µL of 40 mM HYNIC/DMF, 3.23 x 1013 phage pfu, Prep#2: DNase I treatment/purification, 2 hr HYNIC conjugation, 5 µL of 40 mM HYNIC/DMF, 1 x1016 phage pfu, Prep#3: DNase I treatment/purification, 3 min HYNIC conjugation, 20 µL of 40 mM HYNIC/DMF, 8.04 x 1015 phage pfu. In a second set of experiments 20 mg of DNase I was added to 300 to 400 mL of 0.22 µm filtered phagebacteria-LB broth supernatant and incubated at 37 °C for 3 hours prior to further purification, concentration and a 2 hour reaction of 1 x1016 phage in 300 µL of 20 mM HEPES/1x PBS buffer with 5 µL of 40 mM HYNIC/DMF (Prep#2). After a 60 minute radiolabeling reaction with Na (99mTc)pertechnetate and tin-tricine buffer Prep#2 was purified on a NAP-5 desalting column eluted with ten 500 µL fractions of 1x PBS. In contrast to Prep#1, Prep#2 demonstrated a surprising low labeling efficiency of 27.2%, though there was a relatively sharp activity/elution curve with 26.6% added activity seen in the first 5 fractions and 1.3% in the last 5 fractions. 100 uL of the mixture of fractions 3 and 4 was injected into groups of 4 normal mice which were euthanized at 60 minutes for biodistribution assay with results shown in Table 1. There were marked decreases in tracer activity at 60 minutes of Prep#2 in the blood, liver and spleen which were 67%, 83%, and 97% of Prep#1 values, respectively. Prep#2, however, as with Prep#1 showed nearly complete loss of infectivity (< 1% of control). ACS Paragon Plus Environment


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Prep#2 protocol was tested again with shorter HYNIC reaction times of 3 and 30 minutes. While labeling efficiency remained low (31%-34%) the infectivity of phage reacted 3 minutes with HYNIC was greater than 95% of control while a 30 minute reaction with HYNIC resulted in less than 10% infectivity. For Prep#3 bacterial DNA was digested and removed as for Prep#2. However, 8.04 x 1015 phage in 450 µL of 20 mM HEPES/1x PBS buffer was reacted for just 3 minutes with 20 µL of 40 mM HYNIC/DMF. Prep#3 showed a high labeling efficiency of 95% with 94% eluted in the first five 500 µL fractions of 1x PBS eluted on a NAP-5 desalting column. 100 µL of the combined fractions 2 and 3 were injected in 4 normal mice which were euthanized at 60 minutes for biodistribution assay as shown in Table 1. As with Prep#2, Prep#3 showed low tracer activity in the blood, liver and spleen. We confirmed that Prep#3 effectively removed the bacterial contaminants found in Prep#1 with FTIR spectroscopic assay as shown in Table S1. We also confirmed that a HYNIC conjugation time of 3 minutes did not significantly impact the infectivity of Prep#3 phage as compared to unconjugated Prep#3 phage as shown in Table S1. Based on these data the Prep#3 phage purification and HYNIC conjugation protocol (a 3 minute reaction with HYNIC on the day of radiolabeling) was used for all further experiments. The biodistribution of intravenously injected phage has not been exhaustively investigated. An early study reported that

51

Cr-labeled T4 phage immediately clears to the liver25, while only a few years later λ phage

biodistribution was described in Nature as highest in the spleen and to clear much more slowly from that organ than from other tissues26. Our results with Prep#1 and Prep#2 demonstrate that the presence of bacterial DNA not only had a severe adverse impact on the in vivo biostribution of phage but also could not be removed even after multiple washing cycles with 100 kDa and 10 kDa centrifugal filters. The precise reasons behind the adverse effects of bacterial DNA on biodistibution were not studied further in our study but may include; increased phage self-aggregation resulting the formation of colloidal like material prior to injection of labeled tracer, or the unintended conjugation of HYNIC not only to phage but also bacterial DNA or other bacterial contaminants; both factors which promote nonspecific particle uptake by the reticuloendothelial system in the liver and spleen as well as circulating macrophages. Another crucial finding in the biodistribution experiments above was the nearly complete inactivation of highly concentrated purified phage even with dilute concentrations of HYNIC for 2 hours at RT; the recommended time for HYNIC conjugation of large molecular weight proteins31-34. We found that even a brief 30 minute ACS Paragon Plus Environment

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incubation with dilute HYNIC resulted in a more 90% reductivity of infectivity of Pseudomonas bacteriophage as previously described by Cardoso et al.30. The literature has shown that a variety of factors can negatively impact phage infectivity including NaCl concentrations below 5 g/L, the presence of carbohydrates and several amino acids at high concentration (0.5 M), both freeze-thawing and freeze-drying, pH below or above the pH 7 -9 range, AgNO3 (0.5% solution), and Povidone-iodine 1–1.5 mg/L iodine)37-41. These last three factors are particularly problematic for potential phage treatment of skin/superficial wound infections as the pH of skin is roughly 5.5 and infection at these sites are frequently treated with silver nitrate and Povidone-iodine solutions. Prior investigations have also shown that pH below 7 and (close to the pI of a given phage strain) can lead to aggregates which can reach several micrometers in size39 and maybe impart responsible for the non-specific uptake of radiolabeled phage by the reticuloendothelial system noted in previous investigations25-30. Fortunately, we found that a brief 3 minute incubation of purified phage with HYNIC at a molar HYNIC to phage ratio of 100 to 200 followed immediately by quenching with an excess of glycine, resulted in retention of HYNIC-phage infectivity both in vitro and in vivo, without significantly affecting the specific activity of radiolabeled material. Furthermore, we have also placed processed phage prior to HYNIC conjugation into 50 mM Tris, 4g/L NaCl buffer, pH 8.5 at 4 °C for next day use and with 50% glycerol solution at 4 °C for longer term storage. Time Course of Intravenously Injected Radiolabeled Phage Biodistribution Dynamic radionuclide imaging showed a very rapid urinary excretion of tracer (Prep#3) with visible bladder activity occurring within 200 seconds of bolus injection as shown in Figure 3. Total urinary excretion at 5 and 30 minutes was 17.2 ± 5.3% and 31.3 ± 2.7% of Injected Dose (%ID), respectively as shown in Table 2. Urine phage concentrations of >108 PFU/mL (see Table S2) confirm the presence of infective virus though far less than expected based on measurements of excreted

99m

Tc activity. This is likely due to the adverse effects of

low pH on phage infectivity as mentioned above. While significant urinary excretion of tracer was noted previously by Cardosos et al.30 the rapidity of its onset (i.e. starting within 200 seconds after injection) is new. The rapid appearance of activity within the bladder is unlikely to be a result of renal metabolism of labeled bacteriophage or the

99m

Tc-HYNIC moiety; a moiety which when cleaved from labeled proteins within renal

tubular epithelial cells is effectively trapped within the kidney42,43. These data strongly suggest that a conserved ACS Paragon Plus Environment


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mechanism for the rapid and non-inflammatory removal of phage from the blood exists within the mammalian kidney.

Figure 3. Dynamic Imaging Following Bolus Injection of Radiolabeled Phage. Representative dynamic imaging data from a single young adult male mouse immediately following bolus injection of 100 µCi / 3.7 MBq (100 µL) of radiolabeled phage prepared according to the Prep#3 protocol. Activity following intravenous 99mTcHYNIC labeled phage injection is excreted into the bladder within 200 seconds, indicating rapid phage clearance for preparation #3. Mice were sedated with a cocktail of 75 mg/kg and 8 mg/kg of ketamine and xylazine, respectively. Sedated mice were placed in the supine position on top of a high resolution parallel hole collimator (A-SPECT gamma camera) along with a tube containing 1 mL standard (1% of injected activity). Serial 20 second frames were obtained over a period of 30 minutes. Regions of interest (ROIs) were drawn for the left kidney (Lt Kidney), bladder (Bladder), heart and lung (Lung), liver (Liver) and the activity standard. Results are expressed as the %ID per ROI.

Table 2. Carcass and Urinary Activity - Prep#3 %ID Carcass 29 ± 4 30 min 46 ± 21 1 hr 17 ± 4 2 hrs 13 ± 2 3 hrs 15 ± 1 4 hrs

%ID Urine 5 min 17.2 ± 5.3 30 min 31.3 ± 2.7


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Table 2. Total Carcass Activity and Urinary Excretion. Groups of 4 mice underwent biodistribution assay of retained carcass activity at 30 minutes, 1, 2, 3, and 4 hours after intravenous bolus injection of 100 µCi / 3.7 MBq (100 µL) radiolabeled phage prepared according to the Prep#3 protocol. The total amount of urine (before spontaneous voiding) was also collected at 5 and 30 minutes after injection of tracer. The carcass was placed into a clean examination glove after removal of all major organs and urinary bladder and counted in a dose calibrator/well counter (Capintec™) normalized to injected dose assayed separately in a 0.5 mL syringe. Urine was weighed and then counted on a scintillation well counter along with other organ samples. Biodistribution assay at 30 min, 1, 2, 3 and 4 hours revealed a marked wash in and washout of activity in the carcass peaking at 46% of injected activity at 1 hour dropping to 17% at 2 hours (Table 2). There was also a rapid clearance of

99m

Tc-labeled phage from the blood and lungs as shown in Figures 4 and 5 while the uptake

of labeled phage in other major organs including the liver, spleen, kidneys and bone marrow (femur) remained relatively constant after 30 minutes. The marked redistribution of tracer activity in the carcass in normal mice over a period of two hours demonstrates that the majority of intravenously injected phage can rapidly spread throughout the soft tissues of the body without being trapped and is followed by the efficient excretion into the urine when no target bacteria are present. This implies that radiolabeled phage could be highly effective as an imaging agent of soft tissue infection with a rapid wash in and trapping of activity by target bacteria immediately followed by a rapid wash out of unbound activity resulting in a high target to background ratio. In addition, the rapid washout of unbound phage is an advantage in the therapeutic setting in which it is highly desirable to have unused drug or agent be rapidly excreted from the body to decrease treatment related toxicity and the development of potential immune reactions.



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Figure 4. Biodistribution of Intravenously Injected

99m

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Tc-HYNIC-phage in Normal Mice. Groups of 4

normal mice were injected with 100 uCi / 3.7 MBq (100 uL) of radiolabeled phage prepared according to the Prep#3 protocol. Mice were then euthanized at the times listed above and organs and tissues were washed, weighed and counted as described in the Methods section. Results were expressed as the average of the %ID/g of tissue ± one standard deviation. Note that average of renal uptake was divided by a factor of 10 so that these values can be displayed graphically along with the average data from the other organs and tissues of interest. Of the observed organs, the kidneys contained the largest amounts of activity. Activity remained constant over the measured time period for all organs, except blood and lungs.


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Figure 5. Blood Clearance of Radiolabeled Phage. Groups of 4 normal mice were injected with 100 uCi / 3.7 MBq (100 uL) of radiolabeled phage prepared according to the Prep#3 protocol. Mice were then euthanized at the times listed above and blood samples were weighed and counted. Results were expressed as the average of the %ID/g of blood ± one standard deviation. Clearance of blood activity was best described by a biexponential decay curve with a half-life of 26.2 ± 2.6 minutes in the first 30 minutes and 155.7 ± 3.6 minutes between 1 and 3 hours. These data demonstrate the rapid clearance of radiolabeled PAML31-1 from the blood flowing intravenous bolus injection of tracer.

Imaging and Treatment of Lethal Pseudomonas Wound Infection with Radiolabeled Phage To demonstrate in vivo infectivity of 99mTc-HYNIC-modified phage four mice were injected with 20 MBq (250 µL volume, 9.38 x 1014 labeled phage per dose) of radiolabeled phage one day after wounding and inoculation with 1 x 106 Pseudomonas bacteria. 2 hours after injection of tracer sedated mice underwent SPECT imaging as shown in Figure 6.



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Figure 6. SPECT Imaging of Pseudomonas Wound Infection on Day 1 with

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99m

Tc-HYNIC-phage. Four

mice were wounded and infected with 1 x 106 cfus of Pseudomonas aeruginosa strain Xen5 on Day 0. On Day 1 mice were injected with of 20 MBq (550 uCi) of radiolabeled phage (Prep#3) and 2 hours later underwent SPECT imaging. Total SPECT imaging time was just over 30 minutes per animal. After reconstruction image data sets were projected in the sagittal plane and slices summed in 2D for quantification. An upper 5% threshold of activity per pixel was applied to more effectively display wound and lung uptake on each image (i.e. dark liver activity). Total wound uptake was expressed as the ratio of wound related uptake minus the soft tissue background (of the head) divided by soft tissue background. The wound site for each animal is marked with a yellow arrow. Note the multifocal uptake of radiolabeled phage both within the actual wound and the deeper tissues of the back and neck in mouse #1, #3 and #4. In contrast, the mouse #2 had little visible uptake of radiolabeled phage. Wound related uptakes for mouse #1, #2, #3, and #4 were 3.46, 1.36, 15.8, and 5.92, respectively. Despite a single focus of BLI signal (Figure 7A) within the wound SPECT imaging showed extensive multifocal uptake within the deep back fascia and fat, as well as the neck and peri-wound region. The multifocal regional uptake of tracer suggests Pseudomonas bacteria spread to local soft tissues and lymphatic system well beyond that detectable by BLI, though the wound luminescence was roughly proportional to tracer uptake at 24 ACS Paragon Plus Environment

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hours. There was a prompt reduction of BLI signal in all four mice the day after injection of radiolabeled phage which progressively decreased on Days 3 and 4 as shown in Figure 7B.

A)

B) Figures 7A & 7B. BLI Imaging and Serial Monitoring of Wound Infection After Phage Treatment. Representative BLI images of mouse #3 Day 1 and Day 3 after wound infection are shown in Figure 7A. Bacterial burden and luminescence as seen by BLI over time is shown for each infected animal are shown in Figure 7B. Note these four animals also underwent SPECT imaging on Day 1 (shown in Figure 6). Serial BLI ACS Paragon Plus Environment


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imaging demonstrated rapid decrease of bacterial wound burden within 24 hours of a single injection of radiolabeled phage. IVIS imaging of CD1 mice infected with a normally lethal dose of P. aeruginosa showed a marked decrease in bioluminescent signal after phage administration. Radiolabeled phage reduced all wound infections to less than 10% of Day 1 (pretherapy) luminescence by Day 4. All mice survived for another four days without clinical signs of infection. This result is particularly remarkable as bacterial dose in this animal model results in the death of all infected mice within 5 Days44. The doses of bacteria and phage in our wound model are comparable to a recent rabbit model study in which 5 x 106 cfu of methicillin resistant S. aureus were injected into the medullary cavity of the femur followed by four successive doses of 5 x1012 lytic phage injected intraperitoneally at 48 hour intervals starting 16 days after inoculation46. All phage treated rabbits cleared their chronic femoral S. aureus infections by week four without evidence of disease recurrence.

Conclusions The limitations of this study include the labeling of only one phage strain and may limit a broad generalization of the findings, though the similarity of our Prep#1 (non-DNase treated phage) biodistribution data to prior studies suggest that removal of bacterial DNA contaminants from Pbunavirus phages (lytic for Pseudomonas) prior to conjugation with HYNIC is critical. Another inherent set of drawbacks to the direct labeling of parental phage is the inability to image the progeny phage as well as the choice of HYNIC which non-selectively reacts with accessible amine groups possibly comprising phage functionality and biodistribution. Ascertaining the biodistribution of progeny phage is at least as critical to understanding the kinetics/dynamics of phage therapy as that of the parent phage; strategies for identifying the biodistribution of phage progeny include genetic modification using molecular or bioluminescent tagging. Another limitation is that the Pseudomonas phage studied is but one of a number strains, however, we chose this strain given its demonstrated high lytic activity of our selected phage against the clinically relevant Pseudomonas aeruginosa strain Xen5, originally derived from a human septicemia isolate.


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In summary, we describe a new strategy for the radiolabeling of a Pseudomonas phage for biodistibution assay and SPECT radionuclide imaging without adversely impacting its biodistribution or infectivity against a clinically relevant strain of Pseudomonas aeruginosa.

Experimental Procedures Log phase Xen5 cultures P. aeruginosa - Xen05 bioluminescent pathogenic bacteria (#119228, PerkinElmer) was used throughout this study. This product was derived from a human septicemia isolate (American Type Culture Collection, used under license). P. aeruginosa-Xen05 possesses a stable copy of the Photorhabdus luminescens lux operon on the bacterial chromosome. Pseudomonas aeruginosa strain Xen5 was streaked to single colonies on an LB (Luria-Bertani) agar plate, and incubated overnight at 37

0

C. Several colonies (~5) were removed from the

plate using a sterile loop, and cultured overnight, at 370C on a rotator at ~60 rpm, in 2 mL of LB broth. 1 mL of overnight culture was subsequently added to 20 mL of LB broth and incubated at 200 rpm on an orbital shaker, for 3 hours, at 370 C.

PAML-31-1 phage propagation and purification We used the PAML-31-1 (PTA-8362, ATCC) phage which was lytic for the Pseudomonas aeruginosa strain Xen5, throughout this study. 100 uL of log phase Xen5 culture was added to 500 mL of LB broth in a 1L pyrex erlenmeyer flask. 1010 phage (1 uL of a 1:10 dilution of a 1014/mL stock solution) were then added, and the mixture incubated overnight on an orbital shaker at 200 rpm and 37 0C. On the second day, the phage-bacteria-LB broth mixture was transferred to two 500 mL plastic centrifuge bottles with caps, and spun at 4000 rpm for 10 minutes at 4 0C. The supernatant was removed into a new 1L pyrex Erlenmeyer flask, the pellet discarded, and 1 mL of log phase Xen5 culture added (or sufficient log phase culture to ensure the mixture is visibly cloudy). This was again incubated overnight on an orbital shaker at 200 rpm and 37 0C. On the third day, the phage-bacteria-LB broth mixture was again spun at 4000 rpm for 10 minutes at 4 0C. The pellet was discarded, and the supernatant removed and vacuum filtered through a 500 mL 0.22 um filter unit. ACS Paragon Plus Environment


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The resulting stock solution (a) was then stored at 4 0C until ~2 days prior to imaging experiments (storage should not exceed ~1 month). 2 days prior to imaging experiments, the supernatant was retrieved from storage. For Prep#1 the supernatant was then filtered 3 times through Millipore-Amicon Ultra-15 100 kDa 15 mL centrifugal filter units. For Prep#2 and Prep#3 the supernatant was treated with 20 mg of DNase I (MW=40,000, 85365U (dornase units)/mg, Bovine, Pancreas: Millipore/Calbiochem) at 37°C for 3 hours prior to being filtered 3 times through MilliporeAmicon Ultra-15 100 kDa 15 mL centrifugal filter units. For all preparations, each concentration step volume was reduced to

Preparation of Tc99m-Labeled Pseudomonas Bacteriophage without

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